In obesity and type 2 diabetes, Glut4 glucose transporter expression is down-regulated specifically in adipose tissue. We recently determined that adipose-specific down-regulation of Glut4 results in secretion of a novel factor that alters insulin action in muscle and liver. Using DNA array analyses of adipose tissue from adipose-Glut4 knockout and adippse-GLUT4 overexpressing mice, we identified such a protein that is elevated in serum of insulin-resistant rodents and humans, retinol binding protein 4 (RBP4). We demonstrated that RBP4 causes insulin resistance in normal mice. Lowering RBP4 levels in mice that are insulin-resistant due to diet-induced obesity improves their insulin sensitivity. The overall goal of this grant is to determine 1) the physiologic, cellular and molecular mechanisms by which RBP4 causes insulin resistance and 2) whether increased retention of RBP4 is an important mechanism for its serum elevation in insulin resistant states and the mechanisms underlying this. Aim 1 is to determine the physiological mechanisms for the effects of RBP4 on insulin action. We will use in vivo techniques to determine which tissue(s) and physiologic processes underlie the insulin resistance resulting from elevated RBP4 and the ameliorative effect of lowering RBP4 levels in obese and diabetic mice. We will determine whether RBP4 knockout mice are protected from insulin resistance on a high fat diet and the mechanism for the therapeutic effects of a synthetic retinoid that results in RBP4 excretion. Aim 2 is to determine whether the mechanisms by which elevation of RBP4 causes insulin resistance are retinoid-dependent or retinoid-independent. To investigate retinoid-dependent mechanisms, we will measure serum and tissue levels of retinol and retinoids;investigate retinoic acid receptor signaling in obese mice;test the effects of dietary retinol (vitamin A) excess and deficiency on insulin sensitivity;and perform DNA array studies on insulin target tissues of RBP4-injected mice and adipose-Glut4 knockout mice to determine whether the gene expression patterns overlap with a gene set we will establish for retinol excess. To investigate retinol independent mechanisms we will investigate the effects of a mutant RBP4 that does not bind retinol on insulin sensitivity, the metabolic phenotype of mice lacking transthyretin (TTR), and changes in enzymes involved in retinoid metabolism. Aim 3 is to determine whether increased retention of RBP4 is an important mechanism for RBP4 elevation in serum in insulin-resistant states and whether this results from post-translational modifications of RBP4 and/or TTR. Using Mass Spectrometry, we will identify and quantitate TTR protein variants in serum of insulin resistant mouse models and determine whether these are likely to cause RBP4 retention. Since C terminus proteolysis of RBP4 is known to regulate its affinity for TTR, we will determine whether insulin resistance is associated with reduced proteolysis of RBP4 leading to enhanced RBP4 retention in serum. Using purified RBP4 and TTR we will determine which post-translational modifications of TTR alter its binding affinity for RBP4. These studies will elucidate a novel mechanism underlying the inter-tissue communication that plays an important role in the pathogenesis of type 2 diabetes.